The extremely fast rate of enzyme catalyzed reactions makes the direct detection of transient enzyme substrate, E S, complexes a very difficult task. However, the detection of such intermediate states in enzymes is requisite for the ultimate verification of any enzyme mechanism. Therefore, we proposed to develop a new approach to enzyme mechanisms based on direct visualization of these E S complexes by means of Radiationless Energy Transfer (RET). Excitation of enzyme tryptophan allows transfer of energy to the bound dansyl substrate, resulting in reduced enzyme tryptophan fluorescence and enhanced substrate dansyl fluorescence. The energy transfer signal is terminated on dissociation of the dansylated product. Since energy transfer occurs within nanoseconds, changes in dansyl fluorescence accurately monitor both substrate binding and catalysis. The theoretical implications of this approach to enzyme mechanism will first be confirmed by using the class of "seryl" endo-peptidases for which there already exists a large body of information regarding their mechanisms of action. A number of metallo exo- and endo- peptidases and other hydrolytic enzymes for which such information is not available, will then be examined to establish their mechanisms of action. A family of fluorescently labeled substrates have been designed to meet both the enzyme specificity requirements and the spectral characteristics needed for direct observation of E S complexes by radiationless energy transfer. This approach will provide a way to describe the individual steps involved in substrate binding and catalysis and the conformational changes which presumably connect these steps.